Effect of Hemolysis on Laboratory Tests

Hemolysis is the breakdown of red blood cells and the resulting release of hemoglobin, potassium, and other intracellular components into serum or other fluids.  Visually, hemolysis is detected by a characteristic red color in serum or plasma and may be detected by automated serum indices testing by the analyzer. Hemolysis is, in fact, the most common interference seen in serum samples and many laboratory tests may be compromised. Hemolysis occurs in about 3% of all specimens received in the laboratory and accounts for 40% to 70% of all unsuitable specimens. It can occur in vivo (within the body) or in vitro (outside the body).

In vivo hemolysis can be caused by several factors, including infection, disease, trauma, and medications. In vitro hemolysis can be caused by several factors, including improper venipuncture technique, use of tourniquet for too long, centrifugation at too high a speed or too long a time, and shaking or mixing of the sample. Interference may be due to hemoglobin or to other red cell constituents that are released into plasma or serum. These substances can directly or indirectly interfere with several assays, including ALT, AST, creatinine, CK, iron, LDH, lipase, magnesium, phosphorus, potassium, urea, albumin, ALP, chloride, GGT and sodium. For example, hemolysis can cause an increase in the levels of certain enzymes, such as LDH and AST). Hemolysis can also cause a decrease in the levels of certain proteins, such as albumin and hemoglobin. The effects of hemolysis on laboratory tests can vary depending on the type of test and the severity of the hemolysis. In some cases, hemolysis may cause a significant change in the results of a test. In other cases, the effects of hemolysis may be more subtle.

Hemoglobin, which absorbs light strongly at around 550 nm, can cause an apparent increased analyte concentration when monitored near this wavelength. Bichromatic readings and sample blanking will minimize interference; however, the measurement may still be problematic.  Hemoglobin may also have “pseudo” peroxidase activity which may interfere with bilirubin measurement by diazonium methods. Artifactual release of red cell constituents will have obvious effects on the accuracy of serum concentrations, especially K, Mg, P, LDH, AST and ALT.  AST activity is approximately 40-fold higher in erythrocytes than in plasma, so that even slight hemolysis can alter results. Potassium is typically 25 times more concentrated in red blood cells than in plasma. Adenylate kinase released from red cells can increase creatine kinase (CK) activity. Although most phosphate in red blood cells is organic, it can stimulate the release of inorganic phosphate in serum. Hemolysis is a common problem that can affect the results of laboratory tests. It is important to be aware of the causes of hemolysis and the steps that can be taken to prevent it. It is inappropriate to use purified hemoglobin to assess interference from hemolysis because only the direct effect of hemoglobin is evaluated. The effect of dilution and other red cell components should be considered, as well.  CLSI therefore recommends interference testing by spiking hemolysate into a non-hemolyzed serum pool rather than relying on spiking with purified hemoglobin.